A transgenic cell or animal contains one or more transgenes within its genome. A transgene is a DNA sequence integrated at a locus of a genome, wherein the transgenic DNA sequence is not otherwise normally found at that locus in that genome. Transgenes may be made up of heterologous DNA sequences (sequences normally found in the genome of other species) or homologous DNA sequences (sequences derived from the genome of the same species). Transgenic animals have been reported. For example, U.S. Pat. No. 4,736,866 discloses a transgenic mouse containing a c-myc oncogene. Other reports of transgenic animals include PTC Publication No. W082/04443 (rabbit .beta.-globin gene DNA fragment injected into the pronucleus of a mouse zygote); EPO Publication No. 0 264 166 (Hepatitis B surface antigen and Tissue Plasminogen Activator genes under control of the whey acid protein promoter for mammary tissue specific expression); EPO Publication No. 0 247 494 (transgenic mice containing heterologous DNA encoding various forms of insulin); PTC Publication No. W088/00239 (tissue specific expression of DNA encoding factor IX under control of a whey protein promoter); PTC Publication No. W088/01648 (transgenic mammal having mammary secretory cells incorporating a recombinant expression system comprising a mammary lactogen-inducible regulatory region and a structural region encoding a heterologous protein); and EPO Publication No. 0 279 582 (tissue specific expression of chloramphenicol acetyltransferase under control of rat .beta.-casein promoter in transgenic mice).
Transgenic plants have also been produced. For example, U.S. Pat. No. 4,801,540 to Hiatt, et al., discloses the transformation of plant cells with a plant expression vector containing DNA encoding tomato polygalacturonase (PG) oriented in the opposite orientation for expression. The anti-sense RNA expressed from this gene is reportedly capable of hybridizing with the endogenous PG mRNA to suppress translation.
The transgenes introduced into animals and plants so far have been of relatively short length (generally less than about 50 kb). Many eukaryotic genes, however, cover large regions of genomic DNA with many and often very large intervening sequences (introns) between those sequence portions (exons) encoding mRNA. Further, many eukaryotic genes are bounded by regulatory sequences, e.g. enhancers, tissue-specific regulators, cis-acting elements and other physically linked regulatory elements sometimes located many thousands of nucleotides away from the structural gene. The manipulation of such eukaryotic genes has been impeded by their size and complexity. Obstacles include difficulty in the construction, stability, packaging and physical manipulation of large DNA molecules.
Vehicles for cloning DNA have inherent limitations on the size of the DNA they are able to accommodate. Traditional viral vectors such as lambda phage and SV40 have limits of packaging foreign DNA of approximately 50 and 5 kb, respectively. More recently the use of F and P1-based cloning systems, and the cloning of yeast artificial chromosomes have made it possible to propagate larger, contiguous pieces of DNA.
A yeast artificial chromosome or YAC vector, is generated by ligating sequences from a yeast chromosome onto the ends of a piece of DNA. Such sequences include a centromere, two telomeres (one on each end), an origin of replication, and a selectable marker. A telomere is located on each end of the particular piece of DNA to be cloned with a centromere interposed between one of the telomeres and the DNA to be cloned. Several groups have reportedly constructed yeast libraries containing 50-200 kb of human DNA in such YAC vectors (Burke, et al. (1987), Science, 236, 806-812; Traver, et al. (1989), Proc. Natl, Acad. Sci. USA, 86, 5898-5902). Recently, yeast libraries using polyamine condensation to reduce size bias during the yeast transformation step have been reported (McCormick, et al. (1989), Proc. Natl. Acad. Sci. USA, 86, 9991-9995). Libraries produced by this method have an average insert size of 410 kb. Unfortunately, yeast chromosomes are quite difficult and more time consuming to prepare in bulk than are plasmids or vital vectors. Furthermore, there is only one copy of the YAC vector per yeast cell, necessitating the growth of large quantities of the yeast clone for transformations or transfections. Finally, yeast colonies are more laborious to screen than are bacterial colonies and it is not currently possible to ligate such large YAC derived linear DNA segments into circular DNA to transform them into bacteria.
Another method of propagating relatively large DNA molecules involves the use of the P1 cloning system. Sternberg, N. (1990) Proc. Natl. Acad. Sci., USA, 87, 103-107. Vector plasmids for this system contain a P1 packaging site (pac) which is required to initiate the packing of the vector plasmid into the P1 bacteriophage head. The vector plasmid also contains two P1 loxP recombination sites flanking the cloned insert, which are necessary to circularize the packaged linear DNA after P1 phage infection of cells containing the P1 Cre recombinase. The maximum insert size of this system is reported to be about 100 kb because the P1 phage head can accommodate only between 110 and 115 kb of DNA.
Another recently developed method of generating and propagating large pieces of DNA involves the use of F factor-based plasmids. This method reportedly uses sequential homologous recombination steps in E. coli between two closed circular plasmids to sequentially build larger plasmids. Each round of recombination increases the size of the DNA insert in the plasmid. Several plasmids containing 150 kb inserts using this technique have been reported (O'Conner, et al. (1989), Science, 244, 1307-1312). A disadvantage of this approach is that the multi-step procedure is laborious, and requires careful analysis to ensure that rearrangement artifacts have not been introduced. Additionally, even though the technique provides for the production of supercoiled plasmids containing the construct of interest, the large DNA inserts contained therein nevertheless are subject to mechanical shearing when excised. Excision of such inserts is reportedly necessary since plasmid sequences have a negative influence on expression of the transgene (Brinster, et al. (1985), Proc. Natl. Acad. Sci. USA, 4438-4442).
Despite these techniques for propagating larger pieces of DNA, relatively small DNA inserts (e.g. 40-50 kb) continue to be used to transform cultured mammalian cells and to generate transgenic animals.
The invention overcomes the foregoing and other limitations (including the diffiuclty in constructing large DNA molecules) by using homologous recombination in an exquisitely simple way. Although extensive research has been conducted on homologous recombination, the solution to the aforementioned problems in mammalian cell transformation is not apparent from such research.
Gene targeting refers to the directed modification of a selected chromosomal locus of an endogenous chromosome of a cell by homologous recombination with an exogenous DNA sequence having homology to the selected endogenous sequence. Gene targeting has been employed to enhance, modify and disrupt expression of endogenous genes. (See Bollag, et al. (1989), Ann. Rev. Genet., 23,199-225). A significant obstacle to efficient gene targeting in mammalian cells is the ability of these cells to nonhomologously integrate transfected DNA (see Roth and Wilson (1988) In Genetic Recombination, ed. Kucherlapati and Smith, pp. 621-653, Washington, D.C.: Am. Soc. Microbiol.; Brinster, et al. (1985), Proc. Natl. Acad, Sci. USA, 82, 4438-4442). Recently, positive-negative selection vectors have been described for selecting those cells wherein the vector has integrated into a genome by homologous recombination (see e.g. Mansour, S. L., et al. (1988), Nature, 336, 348-352 and Capecchi (1989), Science, 244, 1288-1292). Extrachromosomal homologous recombination refers to homologous recombination occurring within a cell between two exogenous, transfected, and at least partially homologous DNA sequences. This phenomena has reportedly been demonstrated by performing extrachromosomal gene or virus "rescue" experiments (van Zijl et. al (1988), J. Virol., 62, 2191-2195; Miller and Temin (1983), Science, 220, 606-609; Wong and Capecchi (1987), Mol. Cell. Biol., 7, 2294-2295; and for recent review see, Bollag, et al. (1989), Ann. Rev. Genet., 23,199-225). In these studies overlapping pieces of DNA, typically constituting in composite a functional virus, were introduced into cultured cells. Within the cell, the virus sub-fragments were reported to homologously recombine to reconstitute viable virus particles. For instance, van Zijl et. al, transfected, using calcium phosphate precipitation, five overlapping cloned subgenomic fragments of the pseudorabies virus into cultured cells. Some fragments reportedly recombined to form viable virus. Such studies have been directed primarily at manipulating viral genomes, elucidating the mechanisms of and variables affecting efficiency and accuracy of homologous recombination.
Other examples of extrachromosomal homologous recombination involve the introduction of plasmids containing DNA encoding two related but distinct polypeptides in bacterial cell culture. Recombination between the genes encoding such parental polypeptides results in hybrid genes encoding hybrid polypeptides. See e.g. Schneider, W. P. et al. (1981), Proc. Natl. Acad. Sci. USA, 78, 2169-2173 (tryp A); Weber and Weissmann (1983) Nucl. Acids Res., 11, 5661-5669 (alpha interferons); Gray, G. L. et al. (1986), J. Bacteriol., 166, 635-643 (alpha amylases); and EPO Publication No. 0 208 491 published Jan. 14, 1987.
One laboratory has reported extrachromosomal gene rescue in the mouse zygote (Palmiter, et al. (1985) in: Genetic Manipulation of the Early Mammalian Embryo, Cold Spring Harbor Laboratory, p. 123-132). Previously, these investigators reported fusing the mouse metallothionein-I (MT) promoter to the gene for human growth hormone (hGH) (Palmiter, et al. (1983), Science, 222, 809-814) to produce transgenic mice that expressed the MT-hGH transgene and grew larger than control mice. Subsequently, these investigaters reported the construction of two deletion mutants of the MT-hGH DNA transgene (Palmiter, et al. (1985), supra). In one mutant a 5' portion of the MT-hGH DNA segment was deleted. In a second mutant, a portion within the 3' half of the MT-hGH DNA segment was deleted. These investigators reportedly performed a control to a gene rescue experiment by co-injecting the deletion mutants into normal fertilized mouse eggs. Some of the resultant mouse pups reportedly expressed mRNA corresponding to the intact MT-HGH gene and grew larger than their littermates. The mechanism of this rescue remains unclear.
The references discussed above are provided solely for their disclosure prior to the filing date of the present application. Nothing herein is to be construed as an admission that the inventors are not entitled to antedate such disclosure by virtue of prior invention or priority based upon one or more previously filed applications.